SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway.

BACKGROUND: Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms. METHODS: SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of ß-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs. RESULTS: SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear ß-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential. CONCLUSIONS: The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

IGFBP5 enhances osteogenic differentiation potential of periodontal ligament stem cells and Wharton's jelly umbilical cord stem cells, via the JNK and MEK/Erk signalling pathways.

OBJECTIVES: Mesenchymal stem cell (MSC)-mediated tissue regeneration represents a promising strategy for repair of tissue defects, but its molecular mechanisms remain unclear, restricting the use of MSCs. Our previous study indicated that insulin-like growth factor-binding protein 5 (IGFBP5) exerted a valuable effect on osteogenic differentiation of MSCs, but its molecular mechanisms underlying directed differentiation remained unclear. In this study, we have investigated the molecular role of IGFBP5 in regulating this osteogenic differentiation potential. MATERIALS AND METHODS: Periodontal ligament stem cells (PDLSCs) were isolated from periodontal ligament tissue. Wharton's jelly of umbilical cord stem cells (WJCMSCs) was obtained commercially. Lentiviral IGFBP5 shRNA was used to silence IGFBP5. Retroviruses expressing wild-type IGFBP5 were used to overexpress IGFBP5 in the WJCMSCs. Recombinant human IGFBP5 protein (rhIGFBP5) was used to treat PDLSCs for 24 h. Western blot analysis was used to detect the MAPK signalling pathway, and alkaline phosphatase (ALP) activity, Alizarin Red staining and quantitative calcium analysis were used to study osteogenic differentiation potentials. RESULTS: Overexpression of IGFBP5 or rhIGFBP5 increased expression levels of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated mitogen-activated protein kinase 1 and 2 (p-MEK1/2) and phosphorylated extracellular regulated protein kinases (p-Erk1/2) in both WJCMSCs and PDLSCs. Consistently, silenced IGFBP5 was found to effectively inhibit expression of p-JNK, p-Erk1/2 and p-MEK1/2 in PDLSCs and WJCMSCs. Furthermore, inhibition of JNK by its inhibitor, SP600125, or MEK/Erk signalling by its inhibitor, PD98059, dramatically blocked IGFBP5-enhanced ALP activity and in vitro mineralization in both PDLSCs and WJCMSCs. CONCLUSIONS: Our results demonstrated that IGFBP5 promoted osteogenic differentiation potentials of PDLSCs and WJCMSCs via the JNK and MEK/Erk signalling pathways.